1. Field of the Invention
The present invention relates generally to the fields of cancer biology and molecular biology. More particularly, it relates to methods for inhibiting hyperproliferative cell growth and anti-angiogenic effects of C-CAM1.
2. Description of Related Art
Normal tissue homeostasis is achieved by an intricate balance between the rate of cell proliferation and cell death. Disruption of this balance, either by increasing the rate of cell proliferation or decreasing the rate of cell death, can result in the abnormal growth of cells and is thought to be a major event in the development of cancer. The effects of cancer are catastrophic, causing over half a million deaths per year in the United States alone. Conventional strategies for the treatment of cancer, chemotherapy, radiotherapy, surgery, biological therapy or combinations thereof are often ineffective.
In nearly 50% of patients, surgical excision of primary neoplasms is ineffective because metastasis has occurred by the time the tumor is large enough for resection (Sugarbaker, 1977; Fidler and Balch, 1987). Metastases can be located in different organs as well as different regions of the same organ, making complete eradication by surgery, radiation, drugs, or biotherapy difficult. Furthermore, the organ microenvironment significantly influences the response of tumor cells to therapy (Fidler, 1995), as well as the efficiency of anticancer drugs, which must be delivered to tumor foci in amounts sufficient to destroy cells without leading to undesirable side effects (Fidler and Poste, 1985). In addition, the treatment of metastatic cancer is greatly hindered due to the biological heterogeneity of cancer cells, and the rapid emergence of tumor cells that become resistant to most conventional anticancer agents (Fidler and Poste, 1985).
Conventional therapy for malignancy, such as chemotherapy and radiation, has focused on mass cell killing without specific targeting, often resulting in damaging side effects. With advances in molecular genetics and biology, it has become evident that altered expression of normal genes leads to initiation of cancer cells. Cells can be regulated in a positive (stimulatory) or negative (suppressive) manner. Loss of negative regulation of cell growth is often found in malignant cells which exhibit loss of cell proliferation control. Most negative regulators (Marx, 1993; Grunicke and Maly, 1993), referred to as tumor suppressors, have been found to be involved either in direct control of the cell cycle (e.g., Rb, p53, WT-1) or in the signaling pathway leading to cell growth and differentiation (e.g., NF-1).
Thus, the new direction in cancer therapy is to deliver a normal gene to replace or correct the mutated gene, thereby altering the malignant phenotype of transformed cells. However, the transfer of genetic material into cells has limitations. As such, there clearly remains a need for improved methods of anti-hyperproliferative cell therapy.
The present invention addresses the need for improved compositions and methods for anti-hyperproliferative and anti-angiogenic cell therapies. In one embodiment, an objective of the present invention is to provide a composition comprising a cytoplasmic domain of C-CAM1, free from other C-CAM1 domains. In particular embodiments, a composition comprising a cytoplasmic domain of C-CAM1, is provided, wherein the cytoplasmic domain has or comprises the sequence of SEQ ID NO:1. In addition to providing C-CAM1 to a cell or subject, the methods of the invention may further include one or more additional antihyperproliferative or anti-angiogenic therapies, such as surgery, chemotherapy, radiotherapy, hormone therapy, immunotherapy, or gene therapy with other therapeutic genes.
In certain embodiments, a cytoplasmic domain of C-CAM1 further comprises a non-C-CAM1 molecule. In other embodiments, a cytoplasmic domain of C-CAM1 comprises a non-C-CAM1 molecule, wherein the non-C-CAM1 molecule is linked to the C-CAM1 cytoplasmic domain. In additional embodiments, the non-C-CAM1 molecule is selected from the group consisting of a tumor suppressor, an inducer of apoptosis, a cytokine, a targeting sequence, a single chain antibody, an antisense construct, a ribozyme and a chemotherapeutic agent. In one embodiment, the non-C-CAM1 molecule is a tumor suppressor selected from the group consisting of p53, p16, p21, MMAC1, p73, zac1, BRCAI, and Rb. In another embodiment, the non-C-CAM1 molecule is an the inducer of apoptosis selected from the group consisting of Bax, Bak, Bim, Bik, Bid, Bad, Harakiri, Ad E1B, and an ICE-CED3 protease. In yet other embodiments, the non-C-CAM1 molecule is a cytokine selected from the group consisting of IL-2, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, TNF, GMCSF xcex2-interferon, and xcex3-interferon. In still further embodiments, the non-C-CAM1 molecule is a target sequence, wherein the target sequence is a substrate for integrins, proteoglycans, glycoproteins, cell surface receptors, nuclear receptors, or transporters. In a particular embodiment, the non-C-CAM1 molecule is a chemotherapeutic agent, wherein the chemotherapeutic agent is selected from the group consisting of verapamil, podophyllotoxin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, TAXOL (paclitaxel), transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate.
In certain embodiments, the invention involves methods of inhibiting a hyperproliferative cell by administering a C-CAM1 cytoplasmic domain, free from other C-CAM1 domains, to the cell such that the domain inhibits hyperproliferative cell growth. In particular embodiments, the hyperproliferative cell may be a cancer cell, wherein the cancer is selected from the group consisting of lung, breast, melanoma, colon, renal, testicular, ovarian, lung, prostate, hepatic, germ cancer, epithelial, prostate, head and neck, pancreatic cancer, glioblastoma, astrocytoma, oligodendroglioma, ependymomas, neurofibrosarcoma, meningia, liver, spleen, lymph node, small intestine, blood cells, colon, stomach, thyroid, endometrium, prostate, skin, esophagus, bone marrow, and blood. It is contemplated that any of the compounds of the invention may be administered as a pharmaceutical composition to a cell or subject. Components of the composition are pharmaceutically acceptable and are described elsewhere in this disclosure.
In further embodiments, the invention concerns methods of inhibiting a hyperproliferative cell by administering a C-CAM1 cytoplasmic domain, free from other C-CAM1 domains, to the cell, and by also administering a second anti-hyperproliferative agent. The second anti-hyperproliferative agent may be selected from the group consisting of tumor irradiation, chemotherapeutic agent, and a nucleic acid encoding an anti-hyperproliferative polypeptide. In one embodiment, the second anti-hyperproliferative agent is a chemotherapeutic agent selected from the group consisting of verapamil, podophyllotoxin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, TAXOL (paclitaxel), transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate. In another embodiment, the second anti-hyperproliferative agent is radiation, selected from the group consisting of X-ray radiation, UV-radiation, xcex3-radiation, or microwave radiation In a further embodiment, the second anti-hyperproliferative agent is a polypeptide selected from the group consisting of p53, p16, p21, MMAC1, p73, zac1, BRCAI, Rb, Bax, Bak, Bim, Bik, Bid, Bad, Harakiri, Ad E1B, and an ICE-CED3 protease.
In other embodiments of the invention, methods involve a polypeptide that comprises a C-CAM1 cytoplasmic domain, which may be delivered endoscopically, intratracheally, intralesionally, percutaneously, intravenously, aerosolized, intradermally, subcutaneously, intraendothelially, intratumorally, intraperitoneally, intramuscularly, intraendothelially, regionally, locally, or topically.
In another embodiment of the present invention, methods of inhibiting a hyperproliferative cell involve administering an expression construct containing a first C-CAM1 polynucleotide encoding a C-CAM1 cytoplasmic domain, but no other C-CAM1 domains, in an amount effective to inhibit a hyperproliferative cell. The polynucleotide may be linked to a promoter that is operable in eukaryotic cells, which allows the promoter to direct the expression of the C-CAM1 cytoplasmic domain. In particular embodiments, the hyperproliferative cell may be a cancer cell, wherein the cancer is selected from the group consisting of lung, breast, melanoma, colon, renal, testicular, ovarian, lung, prostate, hepatic, germ cancer, epithelial, prostate, head and neck, pancreatic cancer, glioblastoma, astrocytoma, oligodendroglioma, ependymomas, neurofibrosarcoma, meningia, liver, spleen, lymph node, small intestine, blood cells, colon, stomach, thyroid, endometrium, prostate, skin, esophagus, bone marrow, and blood. In other embodiments, inhibiting a hyperproliferative cell by administering an expression construct comprising a first C-CAM1 polynucleotide encoding a C-CAM1 cytoplasmic domain, linked to a promoter operable in eukaryotic cells, further comprises administering a second anti-hyperproliferative agent. In particular embodiments, the anti-hyperproliferative agent is chemotherapeutic DNA damaging agent selected from the group consisting of verapamil, podophyllotoxin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, TAXOL (paclitaxel), transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate. In yet other embodiments, the anti-hyperproliferative agent is tumor irradiation, wherein the radiation is selected from the group consisting of X-ray radiation, UV-radiation, xcex3-radiation, and microwave radiation. In further embodiments, the second polynucleotide encoding a anti-hyperproliferative gene is selected from the group consisting of p53, p16, p21, MMAC1, p73, zac1, BRCAI, Rb, Bax, Bak, Bim, Bik, Bid, Bad, Harakiri, Ad E1B, and an ICE-CED3 protease.
In particular embodiments, the second polynucleotide is operatively linked to a promoter in the first expression construct, wherein the promoter is selected from the group consisting of CMV IE, human or murine MHC class II, SV40, RSV LTR, HIV-1 and HIV-2 LTR. Alternatively, the polynucleotide is operatively linked to a promoter in a second expression construct.
In certain embodiments, the first expression construct is selected from the group consisting of an adenovirus, an adeno-associated virus, a vaccinia virus, and a herpes virus. In yet other embodiments, the first expression construct is non-viral. In other embodiments, the second expression construct is selected from the group consisting of an adenovirus, an adeno-associated virus, a vaccinia virus and a herpes virus, wherein the promoter is selected from the group consisting of CMV IE, human or murine MHC class II, SV40, RSV LTR, HIV-1 and HIV-2 LTR. In yet other embodiments the second expression construct is non-viral. In another embodiment, the administering comprises delivering the expression construct endoscopically, intratracheally, intralesionally, percutaneously, intravenously, aerosolized, intradermally, subcutaneously, intraendothelially, or intratumorally.
In another embodiment of the present invention, methods of inhibiting angiogenesis in a subject involve administering an isolated C-CAM1 polypeptide to a subject in an amount effective to inhibit angiogenesis. It is contemplated that the subject may be a mammal, such as a human. In one embodiment, the C-CAM1 polypeptide is a C-CAM1 cytoplasmic domain free from other C-CAM1 domains. In another embodiment, the C-CAM1 polypeptide is a C-CAM1 cytoplasmic domain free from other C-CAM1 domains wherein the C-CAM1 polypeptide has the sequence of SEQ ID NO:1. In yet another embodiment, administration of a C-CAM1 cytoplasmic domain comprises delivering the polypeptide endoscopically, intratracheally, percutaneously, intravenously, aerosolized, intradermally, subcutaneously, or intraendothelially.
In another embodiment of the invention, a method for inhibiting angiogenesis in a subject comprising the step of administering an expression construct comprising a C-CAM1 polynucleotide encoding a C-CAM1, linked to a promoter operable in eukaryotic cells, wherein the promoter directs the expression of the C-CAM1, which inhibits angiogenesis. In certain embodiments, a C-CAM1 polynucleotide encoding a C-CAM1 encodes a C-CAM1 cytoplasmic domain free from other C-CAM1 domains. In still other embodiments, a C-CAM1 polynucleotide encoding a C-CAM1 encodes a C-CAM1 cytoplasmic domain free from other C-CAM1 domains encodes a C-CAM1 cytoplasmic domain having the sequence of SEQ ID NO:1. In certain embodiments, the expression construct is selected from the group consisting of an adenovirus, an adeno-associated virus, a vaccinia virus and a herpes virus. In other embodiments, the expression construct is non-viral. In yet other embodiments the promoter is selected from the group consisting of CMV IE, human or murine MHC class II, SV40, RSV LTR, HIV-1, and HIV-2 LTR. In another embodiment, administering comprises delivering the expression construct endoscopically, intratracheally, percutaneously, intravenously, aerosolized, intradermally, subcutaneously, intraperitoneally, intramuscularly, intraendothelially, regionally, locally, or topically.
The compositions involved in the methods of the invention may be administered to a cell or subject in an amount effective to render treatment to the cell or subject. In the context of the invention, it is contemplated that the following constitute treatment, though the invention is not limited to these examples: inhibition of angiogenesis, inhibition or suppression of a hyperproliferative cell, which includes inhibiting its growth and or tumorigenicity, as well as inhibition of tumor progression or cancer.
Other methods of the invention include methods of treating a subject with a tumor by giving the patient a composition that contains either (1) an isolated C-CAM1 polypeptide or (2) an expression construct comprising a nucleic acid sequence encoding a C-CAM1 polypeptide under the control of a promoter operable in a eukaryotic cell, in an amount effective to inhibit angiogenesis around the tumor. This may be done in conjunction with tumor resection, which may be performed after or during the treatment with C-CAM-1; alternatively, treatment with C-CAM1 may occur after of all or part of a tumor has been resected, in which case the C-CAM1 is administered to the remaining tumor or tumor bed. Other anti-proliferative treatment may be employed in conjunction with any of the C-CAM1 methods described herein.
Also included are methods that take advantage of any bystander effect of C-CAM1. Therefore, methods for treating a patient with cancer may involve administering to a noncancerous or nonhyperproliferative cell in the patient an effective amount of a composition to confer a therapeutic benefit on the patient. The composition would contain a nucleic acid sequence encoding a C-CAM1 polypeptide under the control of a promoter operable in a eukaryotic cell or a C-CAM1 polypeptide that included the cytoplasmic domain, with or without other C-CAM1 domains. The term xe2x80x9ctherapeutic benefitxe2x80x9d used throughout this application refers to anything that promotes or enhances the well-being of the patient with respect to the medical treatment of his hyperproliferative disease. A list of nonexhaustive examples of this includes extension of the patient""s life by any period of time; decrease or delay in the neoplastic development of the disease; decrease in hyperproliferation; reduction in tumor growth; delay of metastases; reduction in the proliferation rate of a cancer cell, tumor cell, or any other hyperproliferative cell; induction of apoptosis in any treated cell or in any cell affected by a treated cell; and a decrease in pain to the patient that can be attributed to the patient""s condition.
As used in the specification, xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d may mean one or more. As used in the claim(s), the words xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d may mean one or more than one when used in conjunction with the word xe2x80x9ccomprising.xe2x80x9d As used herein xe2x80x9canotherxe2x80x9d may mean at least a second or more.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.